Protein tyrosyl phosphorylation is an important cellular regulatory mechanism. Much work has implicated protein tyrosine kinases (PTKs) in the control of cell proliferation and differentiation. Many PTKs, when mutated and/or captured by retroviruses, can promote oncogenesis (Bishop, J. M., Cell 64:235-248 (1991); Cantley, L. et al., Cell 64:281-302 (1991); Hunter, T., Cell 64:249-270 (1991)). In addition, several PTKs have been shown to be essential for normal differentiation and development. For example, the Drosophila gene torso is essential for proper formation of anterior and posterior structures (Casanova, J. et al., Genes and Devel. 3:2025-2038 (1989)); Sprenger, F., et al., Nature 338:478-483 (1989)). The Caenorhabditis elegans let-23 gene regulates vulval development (Horvits, H. et al., Nature 351:535-541 (1991)), and murine c-kit is required during early embryogenesis for normal hematopoiesis (Geissler, E. N., et al., Cell 55:185-192 (1988); Chabot, B. et al., Nature 335:88-89 1988)).
Much has been learned about PTK signal transduction pathways (Cantley, L., et al., Cell 64:281-302 (1991)). Many growth factor (GF) receptors are transmembrane PTKs, which autophosphorylate upon ligand addition. Activated GF receptors are linked to downstream cellular events by recruitment of secondary signaling molecules to autophosphorylated receptors; some of these signaling molecules are also substrates for the receptor PTKs. Secondary signaling molecules such as GTPase-activating protein (Kaplan, D. R., et al. Cell 61:125-133 (1990)); phospholipase C-.gamma. (Margolis, B., et al., Cell 57:1101-1107 (1989); Meisenhelder, J., et al., Cell 57:1109-1122 (1989)), and phosphatidylinositol 3-kinase (Otso, M., et al. Cell 65:91-104 (1991); Skolnik, E. Y., et al., Cell 65:83-90 (1991); Escobedo, J. A. et al. Cell 65:75-82 (1991)) associate with activated receptors through src-homology 2 (SH2) domains, conserved stretches of approximately 100 amino acids which promote intra- or inter-molecular protein-protein interactions via binding to specific phosphorylated tyrosyl residues (Koch, C., et al., Science 252:668-674 (1991)). Since different subsets of phosphotyrosyl proteins bind to different SH2 domains with varying avidity, the specificity of the cellular response to GFs may be largely determined by the strength and spectrum of these intermolecular SH2/phosphotyrosyl interactions (Koch, C. et al., Science 252:668-674 (1991)).
Src homology region 2 (SH2) is a sequence of about 100 amino acids and was originally identified in the v-Src and v-Fps tyrosine kinases. This noncatalytic domain is conserved among a variety of tyrosine kinases including Src, Src family members, and Fps, for example (Koch, C. A., et al., Science 252:668-674 (1991)). In addition, the SH2 domain is found in the cytoskeletal protein tension, as well as in several cytoplasmic signalling proteins that are regulated by receptor protein-tyrosine kinases, such as phospholipase C-.gamma. and GAP (Ras GTPase activating protein) (Koch, C. A., et al., Science 252:668-674 (1991)).
The SH2 domains present in Src, Abl and Fps tyrosine kinases interact with the kinase domain to modulate activity and may play a role in substrate interaction. In cytoplasmic signalling proteins, the SH2 domain appears to mediate the formation of heteromeric complexes between growth factor receptors and the SH2 domain. In vitro studies have shown bonding of a fragment of GAP containing only the SH2 domains to a C-terminal phosphopeptide from the EGF receptor. It is also though that the SH2 domains of PI3K (phosphatidyl inositol 3'-kinase), GAP and PLC-.gamma. recognize phosphorylated tyrosine on the .beta.-PDGF receptor tyrosine kinase (Koch, C. A., et al., Science 252:668-674 (1991)).
The steady-state level of protein tyrosyl phosphorylation is also controlled by the opposing action of protein tyrosine phosphatases (PTPs); however, little is known about the role of PTPs in signal transduction. Molecular cloning of a large number of PTPs has revealed that they can be grouped into two forms, cytosolic and transmembrane (Fischer, E., et al., Science 253:401-406 (1991)). The cytosolic PTPs include PTP 1B and T cell PTP, both of low molecular weight (37 kD and 48 kD, respectively). These cytosolic PTPs consist primarily of a 300 amino acid domain containing two conserved cysteine active sites with 74% amino acid sequence homology between the two forms. These cytosolic PTPs can be further grouped into subfamilies, based upon distinctive structural features of their non-catalytic regions. These include the presence of a hydrophobic C-terminal sequence in PTP-IB and T-cell PTP (Cool, D. E., et al., Proc. Natl. Acad. Sci. USA 86:5257-5261; Chernoff, J., et al., Proc. Natl. Acad. Sci. USA 87:2735-2739 (1990); Brown-Shimer, S. et al., Proc. Natl. Acad. Sci. USA 87:5148-5152 (1990)), as well as the presence of domains with similarity to cytoskeletal proteins in PTP-Meg (Gu, M. et al., Proc. Natl. Acad. USA 88:5867-5871 (1991)) and PTP-HI (Yang, Q., et al., Proc. Natl. Acad. USA 88:5949-5953 (1991)). Whether these structural similarities have functional significance is not yet known; the regulation and function of non-transmembrane PTPs remain obscure.
The transmembrane PTPs include CD45, also known as leukocyte common antigen (LCA) (Charbonneau, H. et al., Proc. Natl. Acad. Sci. USA 85:7182-7186 (1988)); leukocyte common antigen related protein (LAR) (Streuli, M. et al., J. Exp. Med. 168:1523-1530 (1988)); and two Drosophila homologs (DPTP and DLAR). These transmembrane PTPs are thought to be receptors which modulate their activity in response to ligands as yet unidentified. The transmembrane forms contain a duplication of the 300 amino acid domain present in cytosolic PTPs, creating an imperfect tandem repeat with extensive internal homology between domains I and II. The tandem 300 amino acid domains display homology across forms (to other PTP's of both classes), and across species (human, rat, mouse--90% homology). Of the four conserved cysteines in the 300 amino acid tandem domains (2 per domain), only the first appears essential for phosphatase activity. Two of the cysteines in the first domain represent 2 out of 40 invariant amino acid residues in the first domain of all known PTPs. Although no PTPs have any serine/threonine phosphatase homology, the trans-membrane PTPs possess short hydrophobic transmembrane stretches followed by extracellular regions with immunoglobulin- and fibronectin-like repeats, consistent with ligand binding regions. Despite the sequence conservation in the intracellular (cytoplasmic) domains of the molecules, the extracellular portions have no homology with one another.
Further information concerning PTPs may provide insight into the role of PTPs in cellular function and in regulatory mechanisms in the cell.